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The facile light-driven insertion reaction of oxygen into metal carbon bonds of the BPI (1,3-bis(2-pyridylimino)isoindole) complexes [Pt(BPI)Me] and [Pd(BPI)Me] has been investigated by LED-NMR in CDCl3. The initial insertion reaction leads to peroxo complexes [Pt(BPI)OOMe] and [Pd(BPI)OOMe], which undergo further reactions over time. Spectra were recorded at 1 minute time intervals, which enabled the tracking of the methyl substituent, which eventually generates formaldehyde (and methanediol) and methanol in almost equal proportions. Degradation of the solvent CDCl3 to phosgene and DCl in the presence of oxygen and light leads to several side reactions. DCl reacts with [M(BPI)Me] and [M(BPI)OOMe] to form [M(BPI)Cl], whereas phosgene reacts with in situ generated methanol to chloro methylformate and dimethyl carbonate.

Polycyclic aromatic hydrocarbons (PAHs) are considered priority hazardous substances due to their carcinogenic activity and risk to public health. Strict regulations are in place limiting their release into the environment, but enforcement is hampered by a lack of adequate field-testing procedure, instead relying on sending samples to centralised analytical facilities. Reliably monitoring levels of PAHs in the field is a challenge, owing to the lack of field-deployable analytical methods able to separate, identify, and quantify the complex mixtures in which PAHs are typically observed. Here, we report the development of a hand-portable system based on high-performance liquid chromatography incorporating a spectrally wide absorption detector, capable of fingerprinting PAHs based on their characteristic spectral absorption profiles: identifying 100% of the 24 PAHs tested, including full coverage of the United States Environmental Protection Agency priority pollutant list. We report unsupervised methods to exploit these new capabilities for feature detection and identification, robust enough to detect and classify co-eluting and hidden peaks. Identification is fully independent of their characteristic retention times, mitigating matrix effects which can preclude reliable determination of these analytes in challenging samples. We anticipate the platform to enable more sophisticated analytical measurements, supporting real-time decision making in the field. Portable liquid chromatography instruments enable a myriad of applications in field research. Here a handportable system incorporating a broadband absorption detector is used to separate and classify polycyclic aromatic hydrocarbons from environmental water samples based solely on spectral fingerprinting.

Great strides toward routine single-cell analyses have been made over the last decade, particularly in the field of transcriptomics. For proteomics, amplification is not currently possible and has necessitated the development of ultrasensitive platforms capable of performing such analyses on single cells. These platforms are improving in terms of throughput and multiplexability but still fall short in relation to more established methods such as fluorescence microscopy. However, microscopy methods rely on fluorescence intensity as a proxy for protein abundance and are not currently capable of reporting this in terms of an absolute copy number. Here, a microfluidic implementation of single-molecule microarrays for single-cell analysis is assessed in its ability to calibrate fluorescence microscopy data. We show that the equivalence of measurements of the steady-state distribution of protein abundance to single-molecule microarray data can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy. The methods presented have been developed using GFP but are extendable to other proteins and other biomolecules of interest.

Liquid domains can coexist within lipid membranes, and directly manipulating them in situ may offer a useful tool. Here optical tweezers are used to directly move liquid-ordered domains within the lipid bilayers of individual giant vesicles.

A major trend in analytical chemistry is the miniaturization of laboratory instrumentation. We report a pump requiring no power to operate based on the controlled expansion of a pre-pressurised gas for use in portable applications of high-performance liquid chromatography. The performance of the gas pump is characterised and integrated into a compact liquid chromatography system capable of isocratic separations integrating an LED-based UV-absorption detector. The system weighed 6.7 kg when the mobile phase reservoir was fully charged with 150 mL solvent and included an on-board computer to control the system and analyse data. We characterise the flow-rate through chromatography columns with a variety of geometries and packing materials for a range of pressures up to 150 bar. The maximum variation in flow rate was measured to be 6.5 nL min-1, limited by the resolution of the flow detector. All tests were made on battery power and results are a mixture of those made in the laboratory and in the field. Additionally, we performed a series of 1 m drop tests on the device and show the system's high tolerance to mechanical shocks during operation in the field.

Often cellular behaviour and cellular responses are analysed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behaviour within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumour suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.

For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms are poorly characterized. To further the understanding of these structures, a study of lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy (CLSM). The experimental results confirm that, in accordance with theory, the bilayer interface of a volume asymmetric DIB is curved toward the smaller droplet and a lipid asymmetric DIB is curved toward the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine (PC) are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.

Single molecule microarrays have been used in quantitative proteomics, in particular, single cell analysis requiring high sensitivity and ultra-low limits of detection. In this paper, several image analysis methods are evaluated for their ability to accurately enumerate single molecules bound to a microarray spot. Crucially, protein abundance in single cells can vary significantly and may span several orders of magnitude. This poses a challenge to single molecule image analysis. In order to quantitatively assess the performance of each method, synthetic image datasets are generated with known ground truth whereby the number of single molecules varies over 5 orders of magnitude with a range of signal to noise ratios. Experiments were performed on synthetic datasets whereby the number of single molecules per spot corresponds to realistic single cell distributions whose ground truth summary statistics are known. The methods of image analysis are assessed in their ability to accurately estimate the distribution parameters. It is shown that super-resolution image analysis methods can significantly improve counting accuracy and better cope with single molecule congestion. The results highlight the challenge posed by quantitative single cell analysis and the implications to performing such analyses using microarray based approaches are discussed.

Living cells are hugely complex chemical systems composed of a milieu of distinct chemical species (including DNA, proteins, lipids, and metabolites) interconnected with one another through a vast web of interactions: this complexity renders the study of cell biology in a quantitative and systematic manner a difficult task. There has been an increasing drive towards the utilization of artificial cells as cell mimics to alleviate this, a development that has been aided by recent advances in artificial cell construction. Cell mimics are simplified cell-like structures, composed from the bottom-up with precisely defined and tunable compositions. They allow specific facets of cell biology to be studied in isolation, in a simplified environment where control of variables can be achieved without interference from a living and responsive cell. This mini-review outlines the core principles of this approach and surveys recent key investigations that use cell mimics to address a wide range of biological questions. It will also place the field in the context of emerging trends, discuss the associated limitations, and outline future directions of the field.Impact statementRecent years have seen an increasing drive to construct cell mimics and use them as simplified experimental models to replicate and understand biological phenomena in a well-defined and controlled system. By summarizing the advances in this burgeoning field, and using case studies as a basis for discussion on the limitations and future directions of this approach, it is hoped that this minireview will spur others in the experimental biology community to use artificial cells as simplified models with which to probe biological systems.

We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

Dip-pen nanolithography (DPN) relies on the compatibility between the ink used and the substrate it is written on. This has lead to a significant reliance for DPN on thiol-gold chemistries for the fabrication of nanostructures. This reported work demonstrates a method for creating nanostructures through nanotemplates that allows a wider range of substrates and inks to be used. The substrate was spin-coated with a thin layer of polymer which is selectively removed using a scanning probe microscopy tip coated with a solvent. This produces nanotemplates that are subsequently used to create nanostructures by metallisation. Compared with directly written templates these nanotemplates facilitate longer interaction between inking material and the substrate. They also allow a controlled spatial resolution along the z -axis and eliminate the need for any blocking agents to prevent non-specific adsorption. This method allows DPN to be expanded to applications beyond thiol-gold chemistries.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience. As our understanding of the fundamental mechanistic processes underpinning biology expands, so does the need for high-precision tools to allow the dissection of the heterogeneity and stochastic processes that dominate at the single- and sub-cellular level. Here, we demonstrate a highly controllable and reproducible optical technique for inducing poration within specific regions of a target cell’s plasma membrane, permitting localized delivery of payloads, depolarization and lysis experiments to be conducted in unprecedented detail. Experiments support a novel mechanism for the process, based upon a thermally-induced change triggered by the interactions of a near-IR laser with a biocompatible thin film substrate at powers substantially below that used in standard optoporation experiments.